酒酒球菌环丙烷脂肪酸合酶基因的功能解析

发布时间：2018-03-26 08:44

  本文选题：酒酒球菌　切入点：环丙烷脂肪酸合酶基因　出处：《西北农林科技大学》2017年硕士论文

【摘要】：在葡萄酒酿造过程中,主要利用酒酒球菌(Oenococcus oeni)启动并进行苹果酸-乳酸发酵以改善葡萄酒的风味,而这主要得益于酒酒球菌较强的耐胁迫能力。在酒酒球菌适应胁迫环境过程中,膜成分的自我调节是一个重要机制。菌体主要改变细胞膜的流动性和刚性,以应对外界的胁迫条件,维持细胞正常的生理功能。其中,细胞膜中环丙烷脂肪酸(cyclopropane fatty acid,CFA)的合成在细菌适应急剧变化的环境中非常重要,在酒酒球菌应对多种胁迫环境机制中都发挥着作用。研究和解析环丙烷脂肪酸合酶基因(cyclopropane fatty acid synthase gene,cfa)的功能和菌株耐受性方面的相关性可以更好地探究酒酒球菌应对胁迫环境进行的膜组分变化机制;而植物乳杆菌作为启动苹果酸-乳酸发酵的另一重要菌种,本研究为进一步促进其启动苹果酸-乳酸发酵的广泛实现提供思路和基础。本文首先选择pH作为菌株筛选的胁迫因素,筛选出野生耐酸、酸敏酒酒球菌,确定耐酸野生菌的生长极限。然后,对耐酸、酸敏的野生菌和突变菌进行环丙烷脂肪酸合酶基因进行测序比对。在此基础上,将野生耐酸、酸敏菌株的cfa基因导入大肠杆菌中进行诱导、表达,并获得纯化产物;同时将相同的基因导入植物乳杆菌中进行表达,对重组植物乳杆菌的存活能力、降解苹果酸能力及膜脂肪酸成分变化进行检测。主要完成的研究结果如下:(1)在pH 2.8条件下筛选实验室保藏的35株酒酒球菌,初步筛选出5株较耐酸菌株;在pH 2.6、2.8条件下培养5株菌,测定生存能力(即OD600值)。最终确定酒酒球菌CS-7b及ME-5b的耐酸能力最强,生长极限为pH 2.6。(2)对酒酒球菌CS-7b、ME-5b及酸敏菌株SX-1b与实验室已有突变菌a3(耐酸)及b1(酸敏)进行cfa基因进行测序比对,耐酸菌株的野生型和诱变型的序列相同,较Oenococcus oeni PSU-1发生了3处碱基突变,而酸敏菌野生型、突变型的序列则与Oenococcus oeni PSU-1一致。(3)在大肠杆菌中成功转入SX-1b与CS-7b的cfa基因,构建重组大肠杆菌R1、R2。确定诱导条件,16℃、150 rpm诱导19 h,并过Ni-NTA柱,获得纯化蛋白。(4)将SX-1b与CS-7b的cfa基因在植物乳杆菌ATCC33222中表达,构建重组载体pMG36e-cfa1和pMG36e-cfa2,并得到对应重组菌L1和L2。对植物乳杆菌工程菌的耐酸能力进行检测,并检测胁迫条件下菌体环丙烷脂肪酸含量变化。结果显示在pH 3.6培养基中培养,重组菌L1和L2的生长情况明显优于含空载体的植物乳杆菌L0,且L2的生长优于L1。而在p H 3.4培养基中培养,L0没有生长迹象,只有重组菌L1和L2正常生长,cfa基因的导入突破了植物乳杆菌耐酸的生长极限。在酸胁迫环境中培养重组植物乳杆菌发现,环丙烷脂肪酸的含量急剧上升,且外源cfa基因的导入提升了增幅。L-苹果酸降解试验显示,所有植物乳杆菌工程菌能保持正常苹果酸-乳酸发酵能力。
[Abstract]:In the process of wine brewing, malic acid-lactic acid fermentation was initiated by Oenococcus oeniensis in order to improve the wine flavor. This is mainly due to the strong stress tolerance of Bacillus cereus. The self-regulation of membrane component is an important mechanism in the process of adapting to stress environment. The bacteria mainly change the fluidity and rigidity of cell membrane. The synthesis of cyclopropane fatty acid (Cypropane fatty) on cell membrane is very important for bacteria to adapt to the rapidly changing environment. The study and analysis of the function of cyclopropane fatty acid synthase gene and the relationship between the strain tolerance and the function of cyclopropane fatty acid synthase gene can better explore the relationship between Bacillus cereus and Bacillus cereus. The change mechanism of membrane components in stress environment; Lactobacillus plantarum is another important strain of malolactic acid fermentation. This study provides a basis for further promoting the widespread realization of malic acid-lactic acid fermentation. Firstly, we select pH as the stress factor for screening strains, and select wild acid-resistant, acid-sensitive Bacillus cereus. The growth limit of acid-tolerant wild bacteria was determined. Then, the acid-tolerant, acid-sensitive wild bacteria and mutant bacteria were sequenced and compared with those of cyclopropane fatty acid synthase genes. The cfa gene of the acid-sensitive strain was induced, expressed and purified by E. coli, and the same gene was introduced into Lactobacillus plantarum to express the survival ability of the recombinant Lactobacillus plantarum. The main results were as follows: 1) 35 strains of Bacillus wine were screened under pH 2.8, and 5 strains of acid-resistant strains were screened. Five strains were cultured at pH 2.6 ~ 2.8.The survival ability (i.e. OD600 value) was determined. Finally, the acid-tolerant ability of CS-7b and ME-5b was the strongest. The cfa gene sequence of CS-7bME-5b and acid-sensitive strain SX-1b was compared with that of A3 (acid-tolerant) and b1 (acid-sensitive) strains. The sequence of wild and mutagenic strains was the same. Compared with Oenococcus oeni PSU-1, there were three base mutations, while the wild type of acid-sensitive bacteria, the sequence of which was consistent with that of Oenococcus oeni PSU-1, was successfully transferred into the cfa gene of SX-1b and CS-7b in E. coli. The recombinant Escherichia coli R1 / R2 was constructed. The induction conditions were determined for 19 h after induction at 16 鈩，

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